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Cholesterol- Solubilizing -$gents Related t o the Gallstone Problem
I I 1.-I
CHOLESTEROL-SOLUBILIZIKG AGENTS
Narch 1967
dispersed in human bile is therefore precariously close to crystallization which could occur if any of a number of conditions mere unfavorably altered. Aside from metabolically reducing the amount of cholesterol appearing in the bile, the gallstone problem could most reasonably be approached by attempting to render human bile more capable of holding cholesterol in solution. This might be accomplished by (a) altering biliary pH, ionic environment, protective proteins, and the like to minimize precipitation and concretion; (b) preferentially increasing the concentration of the bile salts, fatty acids, and phospholipids in the bile; or (c) administering an oral agent capable of solubilizing aholesterol in bile to a higher degree than normal. This paper describes our preliminary efforts to find an agent of the type described under (e). The investigation was planned to proceed through ~ o techthree successive stages. First, in ~ i f screening niques would be used to find compounds capable of cholesterol solubilization. Secondly, compounds would be evaluated in vivo to determine biliary excretion following oral administration. Those compounds detected in substantial quantities in the bile of dogs would proceed to the third-stage evaluation. This would be concerned with the relationship of the excreted form of the agent and the cholesterol-carrying capacity of the bile of higher mammals. Methods In Vitro Studies. Test A.-The light-scattering properties of serial dilutions of cholesterol suspensions in solutions containing test compound was measured according to Davis.6 The amount of cholesterol solribilized was obtained from t,he plot of light-scattering us. cholesterol concentration. Test B.-Gallst,ones obt,ained from a single gallbladder and weighing approximately 200 mg each were soaked in water. Each day the gallstone was removed from the water, blotted dry, and weighed in a small covered dish. The procedure was repeated until the weight varied no more than 0.2 mg. Stones prepared in this manner were added to 5 ml of test solution formed from 0.2*5% of test, compound in 0.1 ,!If phosphate buffer a t p H 7.8. The tubes were stoppered and rocked at 25 reciprocations/ miii for 60 hr at 37". The change in weight was recorded. I n V i v o Studies.-Bilefistula rats weighing 450-500 mg were employed. A polyethylene cannula from the proximal portion of the common bile duct was led through a stab wound in the right dorsal side of the body wall. Animals were confined in a Bollman-type restraining cage. The bile was collected continuously in a graduated centrifuge tube. Food and water were given ad libitum. The compounds were administered in a single dose intragastrically, and bile samples were t,aken for 2 days. The apparent concentration of sample was determined by comparison of iiltraviolet spectrophotometric data with that of the administered drrig.6
Results and Discussion Quaternary Nicotinic Acids.-The first' conipounds tested mere a series of alkyl quaternary nicot,inic acids which have the feature of a strong chromophore in the ultraviolet spectral region, greatly aiding in their detection in bile. A literature search revealed that, although many relatively long-chain quaternary salts of nicotinamide,' isonicotinamide,8 ethyl nicotinate,S and ( 5 ) \\.. \V. Davis, Trans. N. Y.A c a d . Sci., 18, 123 (1955). (6) Tile iilriitity of the test compound and the product excreted was not established. (7) XI. F. Zienty, J . A m . Pharm. Assoc., Sci. E d , , 37, 99 (1948). ( 8 ) IT-. Ciusa and 4 . Buccelli, Gazz. Chim. Ital., 88, 393 (1958); Chem. Abstr., 63,20059 (1959). (Y) G. H . Harris, et al., J . A m . Chem. SOC.,73, 3969 (1951).
159
isonicotinates are described, the same is not true for quaternary derivatives of nicotinic and isonicotinic acids. Only three examples of such compounds with chain length greater than C8 (two C16 and one C$ lo were found in the literature. The even-number-carbon chain, decyl to octadecyl quaternary derivatives of nicotinic acid and the dodecyl and tetradecyl derivatives of isonicotinic acid, were prepared in about 50% yield by heating together the acid and alkyl bromide under controlled conditions. In certain examples the bromide salt (infrared absorption at 3.6-4.1 and 5.S p ) was converted to the inner salt (infrared absorption 6.1 p ) by means of a basic, ionCOZH
COzH
R Br-
COzH
R
R C1-
exchange resin. Treatment of t,he inner salt with hydrogen chloride gave the chloride salt. Yields were very low when the chloride salt was prepared directly using alkyl chloride. Sodium 3-pyridinesulfonate reacted with n-decyl and n-dodecyl bromides to form 1-alkyl-3-pyridinium sulfonates" (strong bands at 8.1, 9.6, and 9.7 p ) . The quaternary pyridinium salts that were prepared are listed in Table I. Cholesterol-solubilizing properties of representative members of the series are given in Table 11. Most of these compounds were excreted in the bile of rats. The same compounds, tested in dogs at, a comparable dosage level, were not excreted in the bile. Since the dog has a biliary system more analogous to the human than does the rat', the negative findings in dogs discouraged further invest'igation of this series of compounds. Other Homologous Series.-Bile cont'rast media are usually iodinated phenyl compounds that utilize the radioopaque feature of iodine. l 2 We have synthesized compounds relat'ed to such agents although devoid of the iodine atoms. The modifications brought, together t8heincreased cholesterol-solubilizing feature of a long side chain and the known absorption-excret'ion properties of bile visualization compounds. As an example, Archer and c o - ~ o r k e r prepared s~~ paininobenzenesulfonamides of the type p-IYH2CeHdS02SHCHRC02H in which R was c2-6. We have prepared the higher normal (sahrat'ed) homologs Cg, C1o, C12, and C14 and have noted improved cholest'erol solubilization in vityo with increased chain length. Higher members were not formed under the reaction condit'ions that' gave t'he compounds to CI4. The members of this series and their cholesterol-solubilizing capacit'ies are given in Table 111. The phenoxyalkanoic acid series of t'he t'ype p-RC6H40CRlR2C02H where R is alkyl or acyl and R, and R2 are H or alkyl was prepared and studied for cholesterol solubilizing value. Data are given in Table IV. (10) Alkylation of isonicotinic acid has been reported t o proceed in 1% yield: S.Rlamaliga, Comun. Acad. R e p . Populare Romine, 6, 1583 (1956): Chem. Abstr., SO, 16461 (1966): G . R . Bauxin and F. S.Grossi, U. S. Patent, 2,979,863 (1961): Ciirm. A b s t r . , 65, 27746 (1961). (11) H. hIeyer, .Vonutsli. Cirem.. 24, 195 (1903), reported 1-metliyl-'3pyridinium sulfonate. (12) 9. Archer, Ann. .V. I'.aced. Sei.. 78, 720 (1959). (13) S. Archer, J . 0. Hoppe, T. R. Lewis, and RI. S . Hasliell, J . A7n. I'harm. Assoc., Sci. Ed., 40, 143 (1951).
TABLE IV ~ - ~ C B S T I ' T U T E LPHENOXYACETIC ) ACIDS
R-@Rl~2(~~,),
No.
Ri
Rz
n
Mp ' C
CO,H
Formula
Calcd. R C H
Cl&O4 67.18 114-115 68.16 CljH2004 6849 69.04 C16H2204 93-94 CljH,a04 68.16 112-113 C16H220a 69.04 121-122 69.83 CliH& H 68-69 C&2604 70.56 83-84 CII3 1 109-110 CiiH2404 69.83 H 0 91-92 C14H& 71.15 11 Ci~H2208 71.96 I3 0 39-40 C16H2403 72.69 0 38-39 CllJ C16H2403 72.69 II 0 91-92 €I 73.34 C17H2603 I1 CH3 0 45-46 C1,Hz,OZ 73.93 14 0 35-36 CH3 CII, Aleasrired i n milliglains per milliliier (0.5r; solution of test compound; see Methods in suluble in phosphate buffer. c Tested as the sodium salt. 1% H CH3 H H
1 2 3 4 5 6 7 S 9 IO 11 I:! 1:j
H CHa CH3 H H CH3 CH3 H I1 CII3 CIT3
0 0 0 1 0 0 0
'k
to t,he procedure of Archer.13 The condensation proceeded with imino acids SH2CRIICO,H, where R equals CS, Cl0, (212, and Cl4. \t'7heri higher side-chain amino acids were used under the reported reaction conditions, no reaction took place; when drastic conditions were employed, the corresponding diketopiperazines were formed.': The reported compounds and those of Table I11 showed comparable infrared and ultraviolet spectra. Substituted Phenoxyacetic Acids (Table IV).-4-Acylphenols were prepared according to the procedure of Kindler, et a/.16 These compounds were reduced to the corresponding 4-alkylphenols according to the method of Sandulesco and Girard.I7 The straight-chain and monomethylcarboxylic acids of Table IY were prepared in the follon-ing way. A solution of 0.1 mole of the appropriate phenol, 0.2 mole of KOH, and 200 ml of ethanol was heated to boiling. The appropriate bromocarboxylic acid (0.1 mole), dissolved in 5 0 ml of ethanol, was added dropwise, ailti the resulting solution was heated under reflux for 6 hr. The rexctioii mist lire was cooled, acidified with dilute HCI, and ~~
~-
(15) l'liis stiicly \\ a a rluue by I
